The cell is the most fundamental unit of living organisms, whether animal or plant. The study of its structure and composition, and how its various constituents function, lends valuable insight into the complex processes that occur in integrated biological systems. This requires techniques that allow investigation of cell samples to be conducted in real-time, non-invasively, and in solutions that mimic physiological conditions so that cell functionality is retained.
Optical microscopy (using visible light) has been applied widely to study live cells. However, the resolution is limited by diffraction to about 200-250 nm. For more detailed study, one commonly used method is electron microscopy, where it is possible to obtain images with 10 nm resolution, but the sample needs to be fixed prior to imaging. Hence, it is not possible to use an electron microscope to study living cells.
Another possible high resolution technique is based on the use of scanning probe microscopy (SPM), in which a sharp probe tip is scanned in close proximity to the sample under study. The consequent interactions and thus the chemical/physical properties of the sample can be plotted as a function of the tip's position with respect to the sample, to generate a profile of this measured interaction. Members of the SPM family that are commonly applied to biological imaging are atomic force microscopy (AFM), scanning ion-conductance microscopy (SICM) and scanning near-field optical microscopy (SNOM).
In SICM, an electrolyte-filled, glass micropipette is scanned over the surface of a sample bathed in an electrolytic solution; see Hansma et al (1989) Science 243:641-3. The pipette-sample separation is maintained at a constant value by monitoring the ion-current that flows via the pipette aperture. The flow is between two electrodes: one inside the pipette and another outside in the electrolyte solution. For an applied bias between the electrodes, the ion-current signal depends on a combination of the micropipette's resistance (RP) and the access resistance (RAC) which is the resistance along the convergent paths from the bath to the micropipette opening. RP depends on the tip diameter and cone angle of the micropipette, whereas RAC displays complicated dependence on the sample's electrochemical properties of the bath and the sample, geometry and separation from the probe. It is RAC that lends ion-current sensitivity to the pipette-sample separation and allows its exploitation in maintaining the distance such that contact does not occur.
The optimum tip-sample separation that has allowed SICM to be established as a non-contact profiling method for elaborated surfaces, is approximately one-half of the tip diameter; see Korchev et al (1997), J. Microsc. 188:17-23, and also Biophys. J. 73:653-8. The outputs of the system controlling the position of the tip are used to generate images of topographic features on the sample surface. The spatial resolution achievable using SICM is dependent on the size of the tip aperture, and is typically between 50 nm and 1.5 μm. This produces a corresponding resolution.
However, there is still a need for improved methods and apparatus to study surface structures, e.g. cells.